Wiskott-Aldrich Syndrome (WAS) is an X-linked immunodeficiency caused by mutations that affect the WAS protein (WASP). It is characterized by thrombocytopenia, eczema, impaired immunity and a predisposition to develop lymphomas and leukemias (Cooper, M. D., Chase, H. P., Lowman, J. T., Krivit, W. and Good, R. A. (1968) Am. J. Medicine, 44: 499-513). The size of platelets and lymphocytes is reduced in WAS and scanning electron microscopy of T lymphocytes shows a relatively smooth surface with decrease in the number and size of microvilli, suggesting a defect in cytoskeletal architecture (Remold-O""Donnell, E. and Rosen, F. S. (1993) in Sialophorin (CD43) and the Wiskott-Aldrich Syndrome, eds. Rosen, F. S. and Seligmann, M. S. (Harwood Academic Publishers, Chur), pp. 225-241), pp.225-241 (1993)). The WAS gene is located on Xp11.22-Xp11.23 and encodes a 502 amino acid (a.a.) long proline rich protein, WASP (Derry, J. M. J., Ochs, H. D. and Francke, U. (1994) Cell, 78. 635-644.). WASP contains an N-terminal pleckstrin homology (PH) domain, which partially overlaps with a WASP homology (WH) domain, WH1, found in several proteins involved in the maintenance of cytoskeletal integrity that include Ena, Mena, Evl and VASP (Gertler, F. B., Niebuhr, K., Reinhard, M., Wehiand, J. and Soriano, P. (1996) Cell, 87: 227-239). The WH1 domain in WASP is followed by a GTPase binding domain (GBD/CRIB) (Bunnell, S. C., Henry, P. A., Kolluri, R., Kirchhausen, T., Rickles, R. J. and Berg, L. J. (1996) J. Biol. Chem. 271: 25646-25656), a number of proline rich stretches, a second WH domain (WH2), a short verprolin homology sequence, a cofilin homology sequence, and an acidic C-terminal region. Recently, a protein highly homologous to WASP was cloned from bovine brain and was termed N-WASP (Miki, H., Miura, K. and Takenawa, T. (1996) EMBO J. 15, 5326-5335). N-WASP has a domain organization similar to that of WASP, and is widely expressed, in contrast to WASP which is expressed only in hematopoietic cells.
WASP binds via its GBD domain to the small molecular weight GTPase Cdc42 and weakly to Rac, but not to Rho (Aspenstrom, P., Lindberg, U. and Hall, A. (1996) Curr. Biol. 6: 70-75; Kolluri, R., Tolias, K. F., Carpenter, C. L., Rosen, F. S. and Kirchhausen, T. (1996) Proc. Natl. Acad. Sci. (USA) 93: 5615-5618; Symons, M., Derry, J. M. J., Kariak, B., Jiang, S., Lemahieu, V., McCorrnick, F., Francke, U. and Abo, A. (1996) Cell 84: 723-734). Cdc42, Rac and Rho regulate cytoskeletal organization (Nobes, C. D. and Hall, A. (1995) Cell 81: 53-62). Overexpression of WASP induces the formation of actin-containing clusters (Symons, M., Derry, J. M. J., Kariak, B., Jiang, S., Lemahieu, V., McCorrnick, F., Francke, U. and Abo, A. (1996) Cell 84. 723-734). This is inhibited by dominant negative mutants of Cdc42, but not of Rac or Rho (Symons, M., Derry, J. M. J., Kariak, B., Jiang, S., Lemahieu, V., McCormick, F., Francke, U. and Abo, A. (1996) Cell 84: 723-734). These findings suggest that WASP may provide a link between Cdc42, Rac and the cytoskeleton.
WASP interacts with components of signal transduction pathways via their SH3 domains (Src homology 3) which recognize the proline rich domain in WASP (Featherstone, C. (1997) Science 275: 27-28). WASP associates with the adaptor protein Nck (Rivero-Lezcano, O. M., Marcilla, A., Sameshima, J. H. and Robbins, K. C. (1995) Mol. Cell Biol., 15: 5725-5731). Nck can be recruited via its SH2 domain to tyrosine phosphorylated receptors (Galisteo, M. L., Chernoff, J., Su, Y.-C., Skolnich, E. Y. and Schlessinger, J. (1996) J. Biol. Chem. 271: 20997-21000). WASP also binds in vivo to fyn (Rivero-Lezcano, O. M., Marcilla, A., Sameshima, J. H. and Robbins, K. C. (1995) Mol. Cell Biol. 15: 5725-5731; Banin, S., Truong, O., Katz. D. R., Waterfield, M. D., Brickell, P. M. and Gout, I. (1996) Curr. Biol., 6: 981-988) and in vitro to the src kinase fgr, to the tyrosine kinases btk, itk, Abl and to the p85 subunit of PLC-g (Banin, S., Truong, O., Katz. D. R., Waterfield, M. D., Brickell, P. M. and Gout, I. (1996) Curr. Biol. 6, 981-988; Molina, I. J., Sancho, J., Terhorst, C., Rosen, F. S. and Remold-O""Donnell, E. (1993) J. Immunol., 151. 4383-4390; Finan, P. M., Soames, C. J., Wilson, L., Nelson, D. L., Stewart, D. M., Truong, O., Hsuan, J. J. and Kellie, S. (1996) J. Biol. Chem., 271: 26291-26295).
It would be helpful to have a better understanding of the function of WASP.
Described herein is a novel human gene whose 503 amino acid (a.a.) product interacts with WASP. The protein is referred to as WIP, for WASP-interacting protein. The proline-rich WIP, which co-immunoprecipitated with WASP from lymphocytes, has been shown to bind to WASP at a site distinct from the Cdc42 binding site and to have actin, profilin and Nck binding motifs. Expression of WIP in human B cells, but not of a WIP truncation mutant that lacks the actin binding motif, increased polymerized actin content and induced the appearance of actin-containing cerebriform projections on the cell surface. Work described herein supports the role of WIP in cortical actin assembly that may be important for lymphocyte function. Overexpression of WIP increases F-actin content and induces actin containing structures in the human B cell line BJAB, suggesting an important role for WIP in the organization of the actin cytoskeleton.
In particular, the present invention relates to isolated (e.g., purified, essentially pure) nucleic acids (oligonucleotides, polynucleotides, nucleotide sequences) which encode mammalian (e.g., human) WIP, and include for example, nucleic acids (DNA, RNA) which are obtained from natural sources, recombinantly produced or chemically synthesized. The nucleic acids of the present invention include nucleic acids encoding human WIP (SEQ ID NO: 1) and characteristic portions of the nucleic acid sequences (e.g., probes, primers). The invention also includes complementary sequences (i.e., a complement) of SEQ ID NO: 1 and characteristic portions thereof. The nucleic acids of the present invention encompass nucleic acids encoding a human WIP amino acid sequence (SEQ ID NO: 2) and characteristic portions thereof.
The present invention further relates to isolated, recombinantly produced or synthetic nucleic acids which hybridize to the nucleic acids described herein (e.g., the complement of SEQ ID NO: 1 or characteristic portions thereof) and encode WIP (a protein having the same amino acid sequence as the amino acid sequences included herein and/or a protein which exhibits the same characteristics as WIP described herein). In particular, the invention relates to nucleic acids which hybridize, under moderate or high stringency conditions, to SEQ ID NO: 1 characteristic portions thereof or other sequences which encode WIP.
Also encompassed by the present invention is a nucleic acid construct comprising nucleic acid which encodes a WIP (e.g., SEQ ID NO: 1 and characteristic portions thereof), wherein the nucleic acid of the construct is expressed when the construct is present in an appropriate host cell. In one embodiment, the nucleic acid construct of the present invention is operably linked to exogenous regulatory sequence(s) such as a promoter and/or enhancer, whereby mammalian WIP is expressed hen the host cell is maintained under conditions suitable for expression. The present invention also relates to a host cell comprising nucleic acid encoding mammalian WIP.
Also encompassed by the present invention is a method for producing a WIP (mammalian, such as human). In one embodiment, a nucleic acid construct comprising a nucleotide sequence (DNA, RNA) which encodes a WIP is introduced into a host cell, resulting in production of a recombinant host cell which contains a WIP coding sequence operably linked to an (i.e., at least one) expression control sequence. The host cells produced are maintained in a suitable medium under conditions appropriate for the nucleotide sequence to be expressed, whereby the encoded WIP is produced.
The present invention also relates to isolated (e.g., purified, essentially pure) WIP and includes, for example, WIP obtained from natural sources, recombinantly produced or chemically synthesized. For example, the WIP can be human WIP (SEQ ID NO: 2) or functional portions thereof.
Also encompassed by the present invention is an agent which interacts with WIP directly or indirectly, and inhibits or enhances WIP function. In one embodiment, the agent is an inhibitor which interferes with WIP directly (e.g., by binding WIP) or indirectly (e.g., by blocking the ability of WIP to a) bind WASP, profilin and/or Nck; b) increase F-actin content in lymphocytes; c) increase cellular content of polymerized actin; d) increase appearance of cerebriform projections on the cell surface containing F-actin). In a particular embodiment, an inhibitor of the WIP is an antibody specific for WIP protein or a portion of a WIP; that is, the antibody binds the WIP. For example, the antibody can be specific for the human WIP (SEQ ID NO: 2) or functional portions thereof Alternatively, the inhibitor can be an agent other than an antibody (e.g., small organic molecule, protein, peptide) which binds WIP and blocks its activity. Furthermore, the inhibitor can be an agent which mimics WIP structurally but lacks its function. Alternatively, the inhibitor of WIP can be an agent which binds to or interacts with a molecule which WIP normally binds with or interacts with, thus blocking WIP from doing so and preventing it from exerting the effects it would normally exert. In another embodiment, the agent is an enhancer of WIP which increases the activity of WIP (e.g., the ability of WIP to a) bind WASP, profilin and/or Nck; b) increase F-actin content in lymphocytes; c) increase cellular content of polymerized actin; d) increase appearance of cerebriform projections on the cell surface containing F-actin), increases the length of time it is effective (by preventing its degradation or otherwise prolonging the time during which it is active) or both, either directly or indirectly.
The present invention also relates to antibodies (monoclonal or polyclonal) or functional portions thereof (e.g., an antigen binding portion such as an Fv, Fab, Fabxe2x80x2, or F(abxe2x80x2)2 fragment) which bind WIP.
WIP DNA fragments can be used as probes (e.g., in hybridization methods) or primers (e.g., in amplification methods). They can be used, for example, to determine whether WIP DNA or RNA is present in cells (e.g., a sample obtained from an individual). For example, WIP DNA can be used as a probe to determine if wild-type (nonmutated) or mutated WIP DNA is present in a sample of cells and also to determine the extent (quantity) to which wild-type and mutated forms occur. Antibodies can also be used as probes to assess cells for the occurrence of WIP.
Isolation of WIP makes it possible to detect WIP in a sample (e.g., test sample). The present invention also relates to a method of detecting WIP in a sample (e.g., blood) obtained from an individual, such as a human. In one embodiment, the sample is treated to render nucleic acids in the sample available for hybridization to a nucleic acid probe (e.g., SEQ ID NO: 1 and/or characteristic portions thereof which bind to characteristic regions of WIP-encoding nucleic acids). The treated sample is combined with a nucleic acid probe (labeled or unlabeled) comprising or complementary to all or a characteristic portion of the nucleotide sequence encoding WIP, under conditions appropriate for hybridization of complementary nucleic acids to occur. Hybridization of nucleic acids in the treated sample with the nucleic acid probe is detected; the occurrence of hybridization indicates the presence of WIP in the sample. In another embodiment, the sample is contacted with an antibody which binds to WIP (e.g., SEQ ID NO: 2 or functional portions thereof) under conditions suitable for binding of the antibody to the mammalian WIP. Binding of the antibody to a component of the sample is detected; binding of the antibody to a component of the sample indicates the presence of WIP protein in the sample.
Isolation of WIP also makes it possible to identify a promoter(s) and/or enhancer(s) of the WIP gene. Identification of promoters and/or enhancers of the WIP gene allow for identification of regulators of WIP transcription.
In addition, the present invention relates to transgenic non-human animals (e.g., mice) which lack the WIP gene or contain a nonfunctional WIP gene such that WIP activity is lacking (e.g., WIP knockout mouse). The invention also relates to methods of producing WIP gene knockout animals, such as mice. WIP knockout mice can be used to further study the WIP gene and to assay for inhibitors and enhancers of WIP.
Methods of altering actin content, actin polymerization or both, methods of altering or regulating WASP function and methods of treating conditions in which WIP and/or WASP and/or their respective DNAs are deficient and/or defective are also the subject of this invention. In the methods, WIP or DNA encoding WIP can be administered to an individual, by known methods, in sufficient quantity to alter actin content and/or the extent to which polymerization occurs and, thus, to prevent or reduce (totally or partially) cytoskeletal abnormalities and other adverse effects. For example, Wiskott-Aldrich Syndrome can be treated or prevented in this manner.